Development of human embryonic stem cells (ESCs) and human induced pluripotent stem cells (iPSCs) has unleashed the potential of cellular therapy for a number of human diseases. Replacement therapies using human stem cell-derived ? (SC-?) and SC-myoblasts (the two cell types used in this project), and multiple other stem cell derivatives are of interest to essentially all of the Institutes and Centers at NIH. To avoid rejection, cell replacement therapies will require immune protection by encapsulation, provided immune-evading properties, or given with immunosuppression. As the cell products are of human origin, immunogenicity can't be tested in vivo using immunocompetent animal models due to potent xenograft responses. One can test the cells for safety, efficacy, and immunogenicity in patients, which will be time consuming, expensive, and potentially dangerous. Thus, a critical requirement for the translation of human stem cell-based therapy to the clinic is the development of robust pre-clinical animal models for evaluation of the efficacy, safety and importantly, immunogenicity of human cellular therapy. We propose to modify NOD-scid IL2rgnull (NSG) models of human diabetes and muscle disease to permit development of robust human immune systems. This will allow rapid preclinical evaluation of the function and immunogenicity of human SC-? cells and SC-myoblasts (and SC-derived cells in other models under development). We have documented that current models of NSG mice engrafted with human immune systems cannot fully reject allogeneic human ESCs or SC-endothelial cells, and in preliminary studies, cannot reject human SC-? cells. Our Scientific Premise is that the limited function of the engrafted human immune system in humanized NSG mice is due to lack of organized structure in secondary lymphoid tissues. We have observed that engraftment of newborn NSG mice with wildtype murine HSC will lead to the development of lymph nodes and robust immune responses, suggesting that a lack of species-specific factors impede lymphoid development in humanized NSG mice. In this multi-PI, multi-disciplinary team proposal, Aim 1 will generate novel NSG mouse strains of diabetes and muscular disease that express human specific factors that support lymphoid structure and human immune function. Aim 2 will engraft human SC-? cells and SC- myoblasts into our unique humanized mouse models of diabetes and muscle disease to test for function and immunogenicity. Aim 3 will provide these validated new strains to The Jackson Laboratory (JAX) Biorepository for worldwide distribution. Our proposal takes advantage of powerful new technologies for creating new models of humanized mice, and builds on our track record for generating, validating, and to date, sharing through the JAX distribution network of 17 novel models of humanized NSG mice since 2005. We believe that our innovative approaches, combined with our multi-disciplinary collaborative team will ensure the development of these much-needed preclinical models for regenerative medicine.